September 22, 2010

Jose Ramirez is the Youth Project Coordinator for La Clinica del Pueblo, a non-profit, health center that serves the Latino and immigrant populations of the Washington metro area.

By ANDY WEBSTER
Published: September 16, 2010

Washington has long seemed like the federal government's illegitimate child, denied statehood and degrees of autonomy. Congress's neglect extends to the city's H.I.V. epidemic, a situation made agonizingly clear in Susan Koch's quietly unblinking documentary, "The Other City." The title derives from Washington's split personality: as the illustrious home of politicians and monuments, and as a metropolis with the highest H.I.V. infection rate in the country.

Journalists (Frank Rich of The New York Times, Colbert I. King of The Washington Post); advocates (the writer Larry Kramer); and politicians (Adrian M. Fenty, the mayor of Washington; Representative Eleanor Holmes Norton, a Democrat) offer viewpoints, and the film rolls out disturbing statistics, the most alarming being the city's increase in reported H.I.V.-AIDS cases from 2006 to 2007: 22 percent.

But the most effective voices belong to those with H.I.V., all evincing a visceral sense of mission. J'Mia Edwards is a mother of three struggling to obtain subsidized housing; Ron Daniels is a former drug addict working for a needle-exchange program; JosŽ Ramirez is a gay Latino infected at 17, now dedicated to promoting AIDS awareness among teenagers.

The film's most vivid presence is seen but barely able to speak: Jimmy, a gay man in his 30s dying at Joseph's House, an AIDS hospice. Anyone who has kept a deathbed vigil will relate to his suffering and his family's, and perhaps arrive at a sense of just how universal this epidemic truly is.

THE OTHER CITY

Opens on Friday in New York, Los Angeles and Washington.

Directed by Susan Koch; written by JosŽ Antonio Vargas; director of photography, Neil Barrett; edited by Jeff Werner; music by Charlie Barnett, songs by John Legend; produced by Sheila C. Johnson; released by Cabin Films. In Manhattan at the Chelsea Cinemas, 260 West 23rd Street. Running time: 1 hour 30 minutes. This film is not rated.

In a discovery that sheds new light on the history of AIDS, scientists have found evidence that the ancestor to the virus that causes the disease has been in monkeys and apes for at least 32,000 years - not just a few hundred years, as had been previously thought.

A black colobus monkey from Bioko, one of the species researchers studied to determine the mutation rate of S.I.V.

That means humans have presumably been exposed many times to S.I.V., the simian immunodeficiency virus, because people have been hunting monkeys for millenniums, risking infection every time they butcher one for food.

And that assumption in turn complicates a question that has bedeviled AIDS scientists for years: What happened in Africa in the early 20th century that let a mild monkey disease move into humans, mutate to become highly transmissible and then explode into one of history's great killers, one that has claimed 25 million lives so far?

Among the theories different researchers have put forward are the growth of African cities and the proliferation of cheap syringes.

Confirming that the virus is very old also helps explain why it infects almost all African monkeys but does not sicken them. Over many generations, as any disease kills off vulnerable victims, the host adapts to it.

The new research, published Thursday in Science magazine, was relatively simple. Scientists tested 79 monkeys from Bioko, a volcanic island 19 miles off the West African coast. Bioko used to be the end of a peninsula attached to the mainland in what is now Cameroon, but it was cut off when sea levels rose 10,000 years ago at the end of the last ice age.

Since then, six monkey species have developed in isolation on the island, and scientists from the National Primate Research Center at Tulane University in Louisiana and other American and African universities found that four of them - drills, red-eared guenons, Preuss's guenons and black colobuses - had members that were infected with S.I.V.

The four strains in the four species were genetically very different from one another - meaning they presumably did not come from monkeys carried over to the island by humans in the last few centuries. But each was close to the strain infecting members of the same four genuses on the mainland, meaning they must have existed before Bioko was cut off.

Knowing that all four strains were at least 10,000 years old, scientists recalculated the virus's "molecular clock," measuring how fast it mutates. They now believe that all the S.I.V. strains infecting monkeys and apes across Africa diverged from a common ancestor between 32,000 and 78,000 years ago.

"When we only had 25 years of data, we were dating from the tip at the end of a branch of the evolutionary tree," said Preston A. Marx, a virologist at the Tulane primate center and an author of the paper in Science. "I knew that what we had before couldn't be right, because the virus had spread from the Atlantic to the Indian Ocean to the southern end of the continent, and it couldn't have done that in a couple of hundred years."

Beatrice H. Hahn, a virologist from the University of Alabama at Birmingham and a discoverer of the simian virus, called the study "a very nice paper," adding, "This is what people like us have been looking for."

Previous methods of dating the virus had concluded it was a few hundred to 2,000 years old, "and that just didn't seem right," Dr. Hahn said.

The ancestor virus - which, like many diseases, may have crossed into simians from another, still-unknown species - may have existed for millions of years.

That theory was given greater credence two years ago with the discovery that some Madagascar lemurs have in their genomes the remnants of a virus that was not an S.I.V., but related to it. Madagascar, a Texas-size island 250 miles off the southeastern African coast, separated from Africa 160 million years ago. It has no monkeys, but lemurs' ancestors arrived there, possibly on floating mats of vegetation, probably more than 10 million years ago.

H.I.V., which is almost universally fatal to humans, is obviously very new to us. As Dr. Marx pointed out, if it had been in humans before the 20th century, it would have arrived in the Americas in some of the 12 million Africans kidnapped for the slave trade.

Its immediate ancestor is probably also relatively new to chimpanzees. Last year, Dr. Hahn showed that it can sicken and kill chimps, although not as quickly, meaning they have probably been adapting to it for generations.

The virus has probably crossed over from simians into humans at least five times. There are two human immunodeficiency viruses, H.I.V.-1, by far the most common, and H.I.V.-2, which is milder and rarely seen outside West Africa, and which jumped to humans from sooty mangabeys, a monkey that West Africans hunt and eat.

H.I.V.-1, in turn, has four substrains, designated M, N, O and P. The first, which has spread around the world, clearly came from chimpanzees, as did N and O. But P appears to have crossed over from a gorilla; it was discovered only last year, and in only one woman, who was from Cameroon, where lowland gorillas are hunted for meat.

It is very likely, scientists said, that a little infected monkey or ape blood got into human veins many times in history as hunters cut themselves while butchering carcasses. But even if it sickened those hunters, it probably died out with them or their immediate contacts.

The earliest confirmed H.I.V. case in humans was found in blood drawn in 1959 from a man in Kinshasa, in what was then called the Belgian Congo.

Sometime between the 1800s and 1959, something presumably allowed a human infection with a chimpanzee virus to spread widely enough to evolve into modern H.I.V.-1, which could spread easily among humans.

Dr. Marx believes that the crucial event was the introduction into Africa of millions of inexpensive, mass-produced syringes in the 1950s. Campaigns to wipe out yaws, syphilis, malaria, smallpox and polio required syringes, and many were reused, often with official approval. Traditional healers adopted them for injecting their decoctions, and they became status symbols; a study in Uganda in the 1960s found that 80 percent of families owned one.

Not everyone agrees. Michael Worobey, a virologist at the University of Arizona and another author of the Science paper, said backdating the molecular clock, which he did by comparing the 1959 blood sample with the only other known early one - a paraffin-embedded lymph node from 1960, also from Kinshasa - suggested that the virus emerged closer to 1910, when syringes were handmade, expensive and rare.

He and Dr. Hahn suspect that the growth of colonial cities is to blame. Before 1910, no Central African town had more than 10,000 people. But urban migration rose, increasing sexual contacts and leading to red-light districts.

A study of 107 people coinfected with HCV and HIV found a strong correlation between insulin resistance and both HCV load and sustained virologic response (SVR) to pegylated interferon (PegIFN) alfa2a plus ribavirin [1]. Researchers from the University of Brescia believe their findings help explain the reduced response to PegIFN/ribavirin in coinfected people with insulin resistance.

The study involved 107 HCV/HIV-coinfected people who started PegIFN (180 mcg/week) and ribavirin (1000 to 1200 mg/day) since January 2005. The investigators used the HOMA-IR method to calculate insulin resistance. Study participants had a median age of 43 years (interquartile range [IQR] 40 to 46) and a median HCV load of 5.7 log IU/mL (IQR 4.9 to 6.0). Sixty people (56%) had METAVIR-determined advanced liver fibrosis.

Factors that did not independently affect chances of SVR in this analysis included advanced liver fibrosis, HIV load below 50 copies, total cholesterol, low-density lipoprotein cholesterol, and triglycerides.

"Declining anti-HCV core and envelope-specific antibody responses at the end of therapy were observed only in the SVR group, suggesting that these antibody responses could be used to discriminate between those who experience relapse and SVR at the end of therapy. The SVR group showed the largest and most consistent decrease in titers of antibodies against the core, E1, and NS4 proteins after 48 weeks of treatment. In contrast, the NR and relapse groups showed minimal decreases in antibody titers. Despite the less than detectable levels of HCV RNA at the end of HCV treatment in the relapse and SVR groups, significant decreases in anti-HCV antibody titers do frequently occur among patients with SVRs. Given that HCV loads in the relapse and SVR groups were clinically indistinguishable and below the level of detection, it is possible that the decrease in levels of antibodies in the SVR group reflects a marked decline in antigen load in the liver rather than in the plasma. Regardless of the mechanism, the differential response in antibody titers between the SVR and relapse groups at the end of treatment offers a novel tool to predict who will experience relapse after treatment is stopped. This could lead to the development of novel therapeutic strategies, such as extended therapy for those with relapse. Studies addressing whether these antibodies and/or other biomarkers show robust differences at earlier time points may provide practical tools for monitoring therapy."

"Titers of antibodies against the panel of HCV proteins were also evaluated before and after treatment for their value in monitoring HCV therapy. The Wilcoxon sign ranked test revealed that 4 of the 6 HCV proteins (core, E1, E2, and NS4) showed statistically significant (p<.05) decreases in antibody titer between the pre- and posttreatment samples (Figure 3) . In contrast, antibody responses to the NS3 and NS5A antigens did not significantly change between before and after treatment (p>.44). Substratification by treatment outcome revealed that the SVR group showed the most consistent and largest decrease (p=.02) in titers of antibodies against the 3 most informative antigens (core, E1, and NS4) after treatment (Figure 3) . In contrast, the NR and relapse groups had relatively stable titers of antibodies against these 3 antigens between before and after treatment (p=.70 and p=.43, respectively)"

We quantified antibody responses to the hepatitis C virus (HCV) proteome that are associated with sustained virologic response (SVR) in human immunodeficiency virus (HIV)/HCV-coinfected patients treated with pegylated interferon and ribavirin. Analysis of pre- and posttreatment samples revealed significant decreases in the combined anti-core, anti-E1, and anti-NS4 HCV antibody titers in those with SVRs but not in those who experienced relapse or who did not respond. Furthermore, anti-HIV p24 antibody titers inversely correlated with treatment response. These results suggest that profiling anti-HCV antibody is useful for monitoring HCV therapy, especially in discriminating between those who experience relapse and those who have SVRs at 48 weeks.

Infection with hepatitis C virus (HCV) is seen in 15%-30% of all human immunodeficiency virus (HIV)-infected individuals in the United States, as a result of the shared routes of viral transmission [1, 2]. The introduction of antiretroviral therapy has improved clinical outcomes in patients infected with HIV. However, liver disease has become a leading cause of morbidity and mortality in this population [3, 4]. HIV/HCV coinfection is also associated with higher HCV levels in serum [5, 6], rapid progression of liver disease [7], and lower efficacy of treatment with pegylated interferon plus ribavirin [5, 8]. Development of biomarkers that can accurately predict therapeutic responses are needed to optimize HCV therapy in this coinfected population. Previously, HIV/HCV-coinfected patients who were not responsive to HCV therapy with pegylated interferon plus ribavirin were found to have a gene-activation signature present before treatment indicative of the activation of many immune-related molecules, including interferon-stimulated genes [9]. Quantitative and qualitative humoral responses over the course of HCV therapy among HIV/HCV-coinfected subjects have never been studied, to our knowledge. The ability to clearly predict and monitor outcomes of HCV infection in a robust and simple serological test would have obvious clinical utility. Recently, luciferase immunoprecipitation system (LIPS) assays have been used to accurately quantify antibody responses to various viral pathogens [10]. In the present study, we used LIPS profiling of antibodies against the whole proteome of HCV and part of the proteome of HIV to evaluate its utility in predicting and monitoring the response to HCV therapy in HIV/HCV-coinfected individuals.

Methods.

This was a prospective, open-label trial performed at the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, Bethesda, Maryland. All 29 patients provided written informed consent approved by the NIAID Institutional Review Board. HIV/HCV-coinfected patients were treated with pegylated interferon alfa-2b at 1.5 µg/kg subcutaneously every week (PegIntron; Schering-Plough) and ribavirin daily (Rebetol; Schering-Plough; 400 mg every morning and 600 mg every evening for those <75 kg or 600 mg twice per day for those >75 kg) for 48 weeks and followed up for 24 weeks after the end of treatment. All patients irrespective of virologic response were treated for 48 weeks. One patient discontinued ribavirin at week 24 because of refractory anemia but continued pegylated interferon until week 48.

Patients were eligible for the study if they were >18 years of age and had a CD4 T cell count of >100 cells/µL, an absolute neutrophil count of >1000 cells/µL, an HCV load of >2000 copies/mL, histologic evidence of chronic hepatitis C, and stable HIV disease with or without antiretroviral therapy. Patients with other causes of liver disease, advanced cirrhosis or severe liver decompensation, and several other conditions were excluded. These patients included 11 who experienced no response (NR group), 9 end-of-treatment responders who experienced relapse after 48 weeks of therapy (relapse group), and 9 who experienced sustained virologic responses (SVR group). All patients (except for 1 of the patients in the relapse group who was enrolled in the study and 1 of the previous patients in the NR group who was omitted because of lack of a serum sample) have been described elsewhere [9].

Renilla luciferase (Ruc) antigen fusions-including HCV core, HCV NS3, HCV NS5A, HIV p24 Gag, and HIV Tat-have been described elsewhere [10]. Four additional HCV proteins were generated as Ruc antigen fusions, including E1, E2, NS3, and NS4. One HCV protein, NS5B, was tested, but it was not found to be useful and was not used further. LIPS assays with these different HCV and other Ruc antigens were performed as described elsewhere [11]. All of the light unit (LU) data represent the average of 2 independent experiments and were corrected for background LU values.

Prism software (version 5; GraphPad) was used for statistical analyses. The Mann-Whitney U test was used for comparison of antibody titers between groups, and the Wilcoxon signed rank test was used to evaluate statistical differences between values before and after HCV therapy.

Results.

Antibody titers in serum samples from all patients and in 2 control samples were evaluated for 6 different recombinant HCV antigens, essentially derived from the whole proteome of HCV. A heat map, constructed with log10-transformed antibody titers, was used to display the differing antibody responses to the 6 antigens in individual samples from these subgroups (Figure 1A). As shown by the heat map, LIPS profiling of responses to these 6 HCV antigens clearly distinguished the 29 HCV-infected serum samples from the 2 uninfected control serum samples. The most useful antibody response was directed against the HCV core, for which all but 1 of the 29 HIV/HCV-coinfected samples was positive. The second most useful antibody response was against NS3 (Figure 1A). The other 4 HCV proteins (E1, E2, NS4, and NS5) showed variable immunoreactivity with HIV/HCV-coinfected serum samples (Figure 1A). Of interest, 1 patient in the NR group was completely negative for anti-core, anti-E1, and anti-E2 antibodies but showed strong immunoreactivity to 3 other nonstructural HCV proteins (Figure 1A). Titers of antibodies against the 6 HCV antigens correlated poorly with each other (r8>0.60), suggesting marked heterogeneity in humoral responses (Table 1).

Because of the known effect that HIV/HCV coinfection has on HCV therapy, antibody responses to several HIV proteins were also evaluated in the 3 groups. As shown in Figure 1B, all 29 pretreatment serum samples were robustly seropositive for anti-HIV p24 Gag antibodies by previously determined cutoffs [10], whereas the 2 uninfected control samples were negative. The mean anti-p24 antibody titers in the NR, relapse, and SVR groups were 2.77x10(6) , 2.17x10(6), and 1.71x10(6) LUs, respectively. The anti-p24 antibody titer in the relapse group did not significantly differ from that in either the NR or SVR group (p>.47, Mann-Whitney U test); the NR and SVR groups showed a statistically significant difference in anti-p24 antibody titer (p=0.23). Anti-p24 antibody titers did not correlate (p>.05) with HIV or HCV load, genotype, or CD4 T cell count (data not shown). However, anti-p24 antibody titers paralleled the cumulative group scores for the interferon-associated gene-expression signature previously reported by Lempicki et al [9] for the same patients. Titers of antibodies against the Tat protein of HIV did not significantly differ between the NR, relapse, and SVR groups (p>.26) (data not shown). Because statistically higher anti-p24 antibody titers were detected in the NR group versus the SVR group and correlated with failure of HCV therapy, a cutoff based on 2.2 million LUs was determined to optimally separate these 2 groups. By this approach, 9 of the 11 patients in the NR group were above the cutoff, compared with only 2 of the 9 patients in the SVR group (Figure 1B). On the basis of this analysis, anti-p24 antibody titers provide 82% positive predictive value in identifying patients who will experience therapy failure. Although anti-p24 antibody titers had value only in distinguishing NR from SVR (and none for relapse), there is little practical predictive value for this test in HIV/HCV-coinfected individuals.

Titers of antibodies against the panel of HCV proteins were also evaluated before and after treatment for their value in monitoring HCV therapy. The Wilcoxon sign ranked test revealed that 4 of the 6 HCV proteins (core, E1, E2, and NS4) showed statistically significant (p<.05) decreases in antibody titer between the pre- and posttreatment samples (Figure 3). In contrast, antibody responses to the NS3 and NS5A antigens did not significantly change between before and after treatment (p>.44). Substratification by treatment outcome revealed that the SVR group showed the most consistent and largest decrease (p=.02) in titers of antibodies against the 3 most informative antigens (core, E1, and NS4) after treatment (Figure 3). In contrast, the NR and relapse groups had relatively stable titers of antibodies against these 3 antigens between before and after treatment (p=.70 and p=.43, respectively) (Figure 3). Anti-HIV p24 and anti-BRLF2 Epstein-Barr virus antibody titers also did not change with HCV therapy (data not shown). There was heterogeneity in response to the different HCV antigens in the SVR group, in which some patients showed the largest decrease in anti-HCV core antibody titers, whereas other patients showed more pronounced decreases in anti-ENV1 and anti-NS4 antibody titers. Because decreasing levels of antibodies against these 3 HCV antigens was a common feature of the SVR group versus the NR and relapse groups, the relative decrease in antibody titer between the pre- and posttreatment samples was the most useful approach for distinguishing SVR from NR and relapse. With an antibody titer decrease of >1.5-fold between the pre- and posttreatment samples used as a marker of HCV therapy success, 6 of the 9 patients in the SVR group were positive, compared with only 1 of the 9 patients in the relapse group and none of the patients in the NR group. Overall, this LIPS assay measuring differences in titers of antibodies against these 3 HCV antigens between the pre and posttreatment samples showed an 86% positive predictive value in identifying a response to therapy.

Figure 3. Informative antibody titers before and after hepatitis C virus (HCV) treatment in human immunodeficiency virus-coinfected patients in the no-response (NR; n=11), relapse (n=9), and sustained virologic response (SVR) (n=9) groups. Shown are anti-core, anti-E1, and anti-NS4 antibodies levels at baseline and after treatment in individual patients. The solid horizontal bars reflect the mean titer in each group for the pre- and posttreatment sample. Statistical differences between pre- and posttreatment values were calculated using the nonparametric Wilcoxon signed rank test. The P values derived from summation of the light unit (LU) antibody titers for the 3 antigens are shown at bottom.

Discussion.

Our study suggests that highly quantitative HCV proteome-wide antibody responses can be a valuable tool for monitoring and predicting HCV therapeutic responses among HIV-coinfected patients. Few studies have examined the utility of anti-pathogen antibodies for predicting and monitoring drug therapy. Our LIPS assay provided a clearer summary of the marked patient variability in humoral responses to the whole HCV proteome than has been previously reported. None of the baseline antibody responses to the 6 different HCV proteins predicted response to HCV therapy. This suggests that preexisting host humoral responses to HCV generally do not affect the response to HCV therapy. Previously, it has been shown that, among HCV-monoinfected patients, those with SVRs had higher pretreatment anti-NS4A and anti-NS5a antibody titers (without normalization for HCV load) than did those with NRs [12]. It should be noted that our study differs from this published study in that our patient population was coinfected with HIV and HCV, and HCV loads were controlled for. Nevertheless, 1 patient in the NR group completely lacked anti-core, anti-E1, and anti-E2 antibodies but had high levels of other HCV antibodies, possibly explaining the lack of responsiveness to HCV therapy. Given that this patient (infected with HCV genotype 1) had ample antibodies against HIV and nonstructural HCV proteins, it is likely that selective B cell exhaustion or deletion of certain populations of plasma B cells occurred [13, 14]. Intriguingly, anti-HIV p24 antibodies detected in the pretreatment samples inversely correlated with response to treatment. The highest anti-p24 antibody titers were observed in the NR group, intermediate titers were observed in the relapse group, and the lowest titers were observed in the SVR group. The higher anti-p24 antibody titers in the NR group compared with the SVR group suggests that some of the patients in the NR group who responded poorly to interferon treatment may have had an abnormal immune response to HIV.

Declining anti-HCV core and envelope-specific antibody responses at the end of therapy were observed only in the SVR group, suggesting that these antibody responses could be used to discriminate between those who experience relapse and SVR at the end of therapy. The SVR group showed the largest and most consistent decrease in titers of antibodies against the core, E1, and NS4 proteins after 48 weeks of treatment. In contrast, the NR and relapse groups showed minimal decreases in antibody titers. Despite the less than detectable levels of HCV RNA at the end of HCV treatment in the relapse and SVR groups, significant decreases in anti-HCV antibody titers do frequently occur among patients with SVRs. Given that HCV loads in the relapse and SVR groups were clinically indistinguishable and below the level of detection, it is possible that the decrease in levels of antibodies in the SVR group reflects a marked decline in antigen load in the liver rather than in the plasma. Regardless of the mechanism, the differential response in antibody titers between the SVR and relapse groups at the end of treatment offers a novel tool to predict who will experience relapse after treatment is stopped. This could lead to the development of novel therapeutic strategies, such as extended therapy for those with relapse. Studies addressing whether these antibodies and/or other biomarkers show robust differences at earlier time points may provide practical tools for monitoring therapy.

New Pegasys Patient Assistance Program has generous provisions for patients who want to start HCV therapy with Pegasys and are uninsured. Since Roche & Genentech merged the Genentech Access Solutions P rogram took over this program starting Sept 1 2010 and as I say it is a generous transparent program.

"INNO-LiPA HCV 2.0 currently is the best available commercial assay for HCV genotype 1 subtype identification and should be used in clinical trials and practice.....

Trugene HCV Genotyping Kit and INNO-LiPA HCV 1.0, failed to correctly identify HCV subtype 1a in 22.8% and 29.5% of cases, and HCV subtype 1b in 9.5% and 8.7% of cases, respectively....The second-generation line probe assay is currently the best commercial assay for determination of HCV genotype 1 subtypes 1a and 1b. It can therefore be used locally in clinical trials to identify the HCV subtype and stratify the patients at inclusion, as well as to interpret efficacy and resistance data. When reporting final data, direct sequence analysis of the NS5B region and/or another coding region (for instance the region encoding the antiviral drug target HCV protein) should always be performed as it may identify mistyping or mis-subtyping with commercial assays, especially in the case of rare subtypes."

1 French National Reference Center for Viral Hepatitis B, C and delta, Department of Virology, H™pital Henri Mondor, Universite Paris 12, Creteil, France, 2 INSERM U955, Creteil, France Methods based on the sole analysis of the 5'NCR, namely Trugene HCV Genotyping Kit and INNO-LiPA HCV 1.0, failed to correctly identify HCV subtype 1a in 22.8% and 29.5% of cases, and HCV subtype 1b in 9.5% and 8.7% of cases, respectively (Table 1)...... The results clearly show that, although they are by far the most widely used techniques in new HCV drug development trials, genotyping techniques based on the sole analysis of the 5'NCR should be avoided, as they mistype approximately 25% and 10% of HCV subtype 1a and 1b strains, respectively......INNO-LiPA HCV 2.0 displays the same 5'NCR oligonucleotide probes as INNO-LiPA HCV 1.0, plus core-encoded oligonucleotide probes aimed at better discriminating between HCV subtypes 1a and 1b. With INNO-LiPA HCV 2.0, subtype identification was corrected in 64 of the 70 subtypes 1a that were incorrectly typed with INNO-LiPA HCV 1.0. Five samples could not be PCR-amplified in the core-coding region and the result was not interpretable with INNO-LiPA HCV 2.0 in the remaining case (Table 1). INNO-LiPA HCV 2.0 also corrected subtype identification in 13 of 23 subtypes 1b that were incorrectly typed with INNO-LiPA HCV 1.0. Eight samples could not be PCR-amplified in the core-coding region and the result was not interpretable with INNO-LiPA HCV 2.0 in the remaining two cases (Table 1). Overall, the second-generation line probe assay correctly classified 97.5% of subtype 1a and 96.2% of subtype 1b strains. When only samples that could be PCR-amplified with the assay procedure were taken into account, correct subtype determination was achieved in 99.6% and 99.2% of cases, respectively (Table 1)The real-time PCR-based assay targeting both the 5'NCR and the NS5B region, Abbott RealTime HCV Genotype II assay, correctly identified 93.2% of subtype 1a and 88.9% of subtype 1b strains. Only 2 HCV subtype 1b samples could not be PCR-amplified with this method (Table 1)......Novel assays have been recently developed that aim at better discriminating among the different HCV genotype 1 subtypes and between genotypes 1 and 6. Abbott RealTime HCV Genotype II assay is a real-time PCR method using several sets of genotype- and subtype-specific primers and probes located in both the 5'NCR and the NS5B-coding region. As shown in Table 1, adding a second target region for analysis led to substantially improving HCV genotype 1 subtype identification compared to methods targeting the sole 5'NCR. However, in contrast with a previous report [33], we found that this assay failed to correctly identify HCV genotype 1 subtype in approximately 10% of cases"

Table 1. Ability of the different molecular methods tested in this study to correctly identify HCV subtypes 1a and 1b in a series of 500 patients infected by one or the other of these subtypes.

Abstract

Background

With the development of new specific inhibitors of hepatitis C virus (HCV) enzymes and functions that may yield different antiviral responses and resistance profiles according to the HCV subtype, correct HCV genotype 1 subtype identification is mandatory in clinical trials for stratification and interpretation purposes and will likely become necessary in future clinical practice. The goal of this study was to identify the appropriate molecular tool(s) for accurate HCV genotype 1 subtype determination.

Methodology/Principal Findings

A large cohort of 500 treatment-naïve patients eligible for HCV drug trials and infected with either subtype 1a or 1b was studied. Methods based on the sole analysis of the 5' non-coding region (5'NCR) by sequence analysis or reverse hybridization failed to correctly identify HCV subtype 1a in 22.8%-29.5% of cases, and HCV subtype 1b in 9.5%-8.7% of cases. Natural polymorphisms at positions 107, 204 and/or 243 were responsible for mis-subtyping with these methods. A real-time PCR method using genotype- and subtype-specific primers and probes located in both the 5'NCR and the NS5B-coding region failed to correctly identify HCV genotype 1 subtype in approximately 10% of cases. The second-generation line probe assay, a reverse hybridization assay that uses probes targeting both the 5'NCR and core-coding region, correctly identified HCV subtypes 1a and 1b in more than 99% of cases.

Conclusions/Significance

In the context of new HCV drug development, HCV genotyping methods based on the exclusive analysis of the 5'NCR should be avoided. The second-generation line probe assay is currently the best commercial assay for determination of HCV genotype 1 subtypes 1a and 1b in clinical trials and practice.

Funding: The Trugene HCV 5'NC Genotyping kits and the INNO-LiPA HCV kits were kindly provided by Siemens Medical Solutions Diagnostics. The Abbott RealTime HCV Genotype II kits were kindly provided by Abbott Molecular. This work is part of the activity of the VIRGIL European Network of Excellence on Antiviral Drug Resistance supported by a grant (LSHM-CT-2004-503359) from the Priority 1 "Life Sciences, Genomics and Biotechnology for Health" program in the 6th Framework Program of the European Union. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Introduction

Over 170 million individuals are infected with hepatitis C virus (HCV) worldwide. Phylogenetic analyses have shown that HCV strains can be classified into at least 6 major genotypes (numbered 1 to 6), and a large number of subtypes within each genotype [1]. Genotype 1 is by far the most frequent genotype in chronically infected patients worldwide, with subtypes 1a and 1b representing the vast majority of circulating strains [2], [3], [4].

Current treatment of chronic hepatitis C is based on the combination of pegylated interferon (IFN)-α and ribavirin [5]. This treatment fails to eradicate infection in 50%-60% of patients infected with HCV genotype 1 and approximately 20% of those infected with HCV genotypes 2 and 3 [6], [7], [8]. Thus the need for more efficacious therapies is urgent, especially for patients infected with HCV genotype 1. A number of novel antiviral molecules currently are in preclinical or clinical development [9]. The most advanced ones are specific inhibitors of viral enzymes and functions involved in the HCV life cycle. Molecules that have reached clinical development include inhibitors of the nonstructural (NS) 3/4A serine protease and inhibitors of HCV replication that belong to different categories: nucleoside/nucleotide analogue and non-nucleoside inhibitors of the HCV RNA-dependent RNA polymerase (RdRp), NS5A inhibitors and cyclophilin inhibitors [9]. These agents have shown potent antiviral efficacy when used alone, and encouraging results have been recently published showing that HCV clearance can be achieved in approximately 70% of cases when a potent NS3/4A inhibitor is used in combination with pegylated IFN-α and ribavirin [10], [11], [12].

HCV genotype 1 is generally considered as a homogeneous group. There are however biological differences between the different subtypes of HCV genotype 1, which are related to differences in their nucleotide and amino acid sequences. Importantly, differences between subtype 1a and 1b (by far the most frequently encountered genotype 1 subtypes in clinical practice) include different efficacies of antiviral drugs and different resistance profiles to such drugs. Indeed, several HCV inhibitors appear to have selective activity against different HCV genotype 1 subtypes, both in vitro and in vivo. Differences have been observed in vitro with NS3/4A protease inhibitors, non-nucleoside inhibitors of HCV RdRp and NS5A inhibitors [13], [14], [15], [16], [17]. For instance, BILB 1941, a non-nucleoside inhibitor of HCV RdRp, has been shown to have better antiviral efficacy in patients infected with HCV subtype 1b than in those infected with HCV subtype 1a, a finding reflecting in vitro experiments [13].

A major issue that limits the efficacy of direct acting antiviral therapies for HCV is the selection by these drugs of resistant variants upon administration [18]. Recent studies with NS3/4A protease inhibitors have shown that the genetic barrier and resistance profiles substantially differ between the different genotype 1 subtypes. For instance, the Arg to Lys substitution at position 155 of the NS3 protease (R155K) is usually selected in subtype 1a replicons treated with telaprevir, but not in subtype 1b replicons [19]. The reason is that only one nucleotide substitution is needed relative to the subtype 1a sequence to generate this variant, whereas two substitutions are needed relative to the 1b sequence (codon usage bias). Overall, natural polymorphisms at positions R155 and V36 are frequent in subtype 1a, but rare in subtype 1b where substitutions at position A156 are preferentially selected in vitro [19]. This is reflected in vivo by the different resistance profiles in patients infected by HCV subtypes 1a and 1b. In the former, the V36 and R155 substitutions represent the backbone of resistance, whereas in the latter resistance is less frequent as it is preferentially associated with substitutions at position A156 that are associated with a decreased fitness of the variants [19], [20], [21]. Similarly, important differences in the resistance profiles have been described in vitro with HCV-796, a non-nucleoside inhibitor of HCV RdRp. The C316Y amino acid substitution has been reported to be selected in both subtype 1a and 1b replicon cells. However, in genotype 1a replicons, the C316Y substitution has low replication capacity that must be compensated for by additional "compensatory" substitutions, including L392F or M414T, resulting in an increase in replication levels of at least 10-fold [19]. A higher genetic barrier to resistance to HCV-796 and related compounds is therefore expected in patients infected with HCV subtype 1a than 1b. In vivo, HCV-796 monotherapy was however shown to select subtype 1a variants with a single C316Y substitution, whereas the C316Y substitution was associated with a number of additional substitutions in subtype 1b patients [22].

As a result of these findings, correct identification of HCV subtypes 1a and 1b is crucial in clinical trials assessing new HCV drugs in order to correctly stratify and interpret efficacy and resistance data. It may also become important in future clinical practice, as tailoring treatment schedules with HCV inhibitors to HCV genotype 1 subtype might become necessary. A variety of molecular methods can be used to identify the HCV genotype and subtype both in clinical trials and practice. Commercial assays have been developed, most of them targeting the 5' noncoding region (5'NCR) of the HCV genome, although this region is the most conserved one. These methods have been shown to differentiate well the different HCV genotypes (1 to 6), except genotype 1 from genotype 6, a rare HCV genotype in the Western world [23], [24]. The goal of our study was to assess the ability of molecular methods targeting the 5'NCR to correctly identify the HCV genotype 1 subtype in patients eligible for clinical trials, and to identify the best method for this purpose.

Results

Hepatitis C Virus Genotype and Subtype Determination by Phylogenetic Analysis of a Portion of the NS5B Gene

Direct sequence analysis of a sufficiently long portion of the NS5B gene followed by phylogenetic analysis is the reference method for identification of HCV genotype and subtype [1], [25]. It was used to identify the HCV genotype and subtype in 516 treatment-naïve patients included in a multicenter clinical trial assessing different schedules of pegylated IFN-α2a and ribavirin [26]. All of these patients were thought to be infected with HCV genotype 1 at inclusion based on local assessment. In fact, 6 patients were infected with genotype 6, including 2 with subtype 6e, one with subtype 6o, one with subtype 6p, one with subtype 6q and one with subtype 6r. These 6 samples were not considered for further analysis in the present study. The remaining 510 patients were confirmed to be infected with HCV genotype 1: 237 of them (46.5%) were infected with HCV subtype 1a and 263 (51.6%) with subtype 1b (Figure 1). As shown in Figure 1, HCV subtype 1a strains segregated into two distinct clades, that were termed 1a clade I (n = 83, 35.0%) and 1a clade II (n = 154, 65.0%). Eight patients (1.6%) were infected with another HCV genotype 1 subtype, including 4 patients with subtype 1d, 2 with subtype 1e, one with subtype 1i, and one with subtype 1l. The remaining 2 patients (0.3%) were infected with genotype 1 but the subtype could not be determined. The ability of the different molecular methods to correctly identify HCV subtypes 1a and 1b was then tested on the 237 and 263 samples containing HCV subtypes 1a and 1b, respectively.

INNO-LiPA HCV 2.0 displays the same 5'NCR oligonucleotide probes as INNO-LiPA HCV 1.0, plus core-encoded oligonucleotide probes aimed at better discriminating between HCV subtypes 1a and 1b. With INNO-LiPA HCV 2.0, subtype identification was corrected in 64 of the 70 subtypes 1a that were incorrectly typed with INNO-LiPA HCV 1.0. Five samples could not be PCR-amplified in the core-coding region and the result was not interpretable with INNO-LiPA HCV 2.0 in the remaining case (Table 1). INNO-LiPA HCV 2.0 also corrected subtype identification in 13 of 23 subtypes 1b that were incorrectly typed with INNO-LiPA HCV 1.0. Eight samples could not be PCR-amplified in the core-coding region and the result was not interpretable with INNO-LiPA HCV 2.0 in the remaining two cases (Table 1). Overall, the second-generation line probe assay correctly classified 97.5% of subtype 1a and 96.2% of subtype 1b strains. When only samples that could be PCR-amplified with the assay procedure were taken into account, correct subtype determination was achieved in 99.6% and 99.2% of cases, respectively (Table 1).

The real-time PCR-based assay targeting both the 5'NCR and the NS5B region, Abbott RealTime HCV Genotype II assay, correctly identified 93.2% of subtype 1a and 88.9% of subtype 1b strains. Only 2 HCV subtype 1b samples could not be PCR-amplified with this method (Table 1).

5'NCR Sequence Analysis in Misclassified Subtype 1a Strains

Among the HCV subtype 1a strains, 47 were misclassified as subtype 1b by Trugene HCV Genotyping Kit and/or INNO-LiPA HCV 1.0, including 33 that were misclassified by both assays, 7 that were misclassified by Trugene HCV Genotyping Kit only, and 7 that were misclassified by INNO-LiPA HCV 1.0 only (Table 2). Figure 2 shows an alignment of their 5'NCR sequences relative to the consensus sequences of the correctly classified strains (including subtype 1a clade I, subtype 1a clade II and subtype 1b). As shown in Figure 2, misclassification of subtype 1a strains into subtype 1b in one or both assays was related to the presence of natural polymorphisms at nucleotide positions 204 and 243, both of which are located within the sequence of an INNO-LiPA HCV 1.0 probe. At position 243, A is the most frequent nucleotide in HCV subtype 1a, in both subtype 1a clade I and clade II. Substitution into a G, the most frequent nucleotide at position 243 in subtype 1b, was found in all cases that were misclassified as subtype 1b by Trugene HCV Genotyping Kit and/or INNO-LiPA HCV 1.0 (Figure 2). At position 204, A is the most frequent nucleotide for subtype 1a clade I, whereas C is the most frequent nucleotide for subtype 1a clade II, and C or T are the most frequent nucleotides for subtype 1b. In spite of the presence of a G at position 243, the presence of an A at position 204 allowed correct identification of subtype 1a with Trugene HCV Genotyping Kit but not with INNO-LiPA HCV 1.0 (Figure 2). The usual presence of a C at position 204 in subtype 1a clade II explains why misclassifications were far more frequent with this clade than with subtype 1a clade I.

Among the 12 subtype 1a strains that were classified as genotype 1, indeterminate subtype with Trugene HCV Genotyping Kit, one had a G and 5 had mixed A and G populations at position 243. Two additional patients with an A at position 243 had a C at position 248. In the remaining 4 cases, no explanation was found in the 5'NCR sequence for the failure to identify the HCV subtype (data not shown). Among the 25 subtype 1a strains that were classified as genotype 1, indeterminate subtype with INNO-LiPA HCV 1.0 (including 6 with the same profile in Trugene HCV Genotyping Kit), 4 had a G and 4 had mixed A and G populations at position 243. Three additional patients with an A at position 243 had a C at position 248 (C only in two of them, a mixture of C and T in one). In the 14 remaining cases, no explanation was found in the 5'NCR sequence for the failure to identify the HCV subtype (data not shown).

5'NCR Sequence Analysis in Misclassified Subtype 1b Strains

Among HCV subtype 1b strains, 8 were misclassified as subtype 1a by Trugene HCV Genotyping Kit and/or INNO-LiPA HCV 1.0, including 3 that were misclassified by both assays, 4 that were misclassified by Trugene HCV Genotyping Kit only, and 1 that was misclassified by INNO-LiPA HCV 1.0 only (Table 2). Figure 3 shows an alignment of their 5'NCR sequences relative to the consensus sequences of the correctly classified subtype 1a and subtype 1b strains. As shown in Figure 3, and as for misclassified subtype 1a strains discussed above, misclassification of subtype 1b strains into subtype 1a was related to the presence of natural polymorphisms at positions 204 and 243. At position 243, G is the most frequent nucleotide in HCV subtype 1b. Substitution into an A, the most frequent nucleotide at position 243 in subtype 1a, was found in all cases that were misclassified as subtype 1a by both Trugene HCV Genotyping Kit and INNO-LiPA HCV 1.0 and by INNO-LiPA HCV 1.0 only, but not in those that were misclassified by Trugene HCV Genotyping Kit only (Figure 3). In the latter, it is the presence of an A at position 204 instead of a C or a T that was responsible for misclassification in all but one case (Figure 3).

Among the 11 subtype 1b strains that were classified as genotype 1, indeterminate subtype with Trugene HCV Genotyping Kit, one had an A at position 243. In the remaining cases, no explanation was found in the 5'NCR sequence for the failure to identify the HCV subtype (data not shown). Among the 15 subtype 1b strains that were classified as genotype 1, indeterminate subtype with INNO-LiPA HCV 1.0 (none of which were classified as indeterminate in Trugene HCV Genotyping Kit), one had an A and one harbored mixed A and G populations at position 243. Both of them had a C at position 248 (C only in one of them and a mixture of C and T in the other one). In the remaining 13 cases, no explanation was found in the 5'NCR sequence for the failure to identify the HCV subtype (data not shown).

Among the HCV subtype 1a strains, 16 were incorrectly classified by Abbott RealTime HCV Genotype II assay (Table 1): 2 were misclassified as subtype 1b, 12 were classified as genotype 1, indeterminate subtype, one was identified as a mixed 1a/1b infection, and one gave an indeterminate result. In one case, PCR amplification failed, and in one case, not enough serum volume was available for testing.

Among the HCV subtype 1b strains, 27 were incorrectly classified by Abbott RealTime HCV Genotype II assay (Table 1): 3 were misclassified as subtype 1a, 18 were classified as genotype 1, indeterminate subtype, 5 were identified as a mixed 1a/1b infection, and one gave an indeterminate result. In 2 cases, PCR amplification failed, and in one case, not enough serum volume was available for testing.

"The reliability of genotyping methods highly depends on the amount of information (i.e., the number of informative sites) that is utilized for the discrimination of genetic variants......Our results indicate that Versant HCV genotype assay (LiPA) 2.0 yielded an interpretable genotype result for 96.0% of the samples and that 99.4% of the interpretable results agreed with the reference method, rendering it an accurate and reliable assay suitable for large-scale genotyping. This new assay outperforms the previous version of the line probe assay, since Versant HCV genotype assay (LiPA) 1.0 has an overall accuracy of 74%, taking subtype i nformation into account (8, 23).....In conclusion, Versant HCV genotype assay (LiPA) 2.0 provides a rapid, sensitive, and accurate means of HCV genotyping and can be used as a routine tool to distinguish between the different HCV genotypes and subtypes. Considering the importance of genotype determination in understanding the epidemiology of the virus and in the management of hepatitis C treatment strategies, efficient genotyping tools are indispensable in clinical diagnostic settings."

Hepatitis C virus (HCV) genotyping is a tool used to optimize antiviral treatment regimens. The newly developed Versant HCV genotype assay (LiPA) 2.0 uses sequence information from both the 5' untranslated region and the core region, allowing distinction between HCV genotype 1 and subtypes c to l of genotype 6 and between subtypes a and b of genotype 1. HCV-positive samples were genotyped manually using the Versant HCV genotype assay (LiPA) 2.0 system according to the manufacturer's instructions. For the comparison study, Versant HCV genotype assay (LiPA) 1.0 was used. In this study, 99.7% of the samples could be amplified, the genotype of 96.0% of samples could be determined, and the agreement with the reference method was 99.4% when a genotype was determined. The reproducibility study showed no significant differences in performance across sites (P = 0.43) or across lots (P = 0.88). In the comparison stud y, 13 samples that were uninterpretable or incorrectly genotyped with Versant HCV genotype assay (LiPA) 1.0 were correctly genotyped by Versant HCV genotype assay (LiPA) 2.0. Versant HCV genotype assay (LiPA) 2.0 is a sensitive, accurate, and reliable assay for HCV genotyping. The inclusion of the core region probes in Versant HCV genotype assay (LiPA) 2.0 results in a genotyping success rate higher than that of the current Versant HCV genotype assay (LiPA) 1.0.

INTRODUCTION

Hepatitis C virus (HCV) is a leading cause of chronic liver disease and has already infected at least 170 million people worldwide. Each year, 3 to 4 million people are newly infected. HCV creates an extensive disease burden, since it accounts for 20 to 30% of cases of acute hepatitis, 70 to 80% of cases of chronic hepatitis, 40% of cases of end-stage cirrhosis, 50 to 76% of cases of hepatocellular carcinoma, and 30 to 40% of liver transplants (15, 33, 34).

HCV belongs to the family of the Flaviviridae and can be divided into different genotypes based on phylogenetic analysis of full-length or partial sequences of HCV strains. The most current consensus proposal distinguishes six genotypes based on phylogenetic cluster analysis of complete genomes. The genotype formerly designated as 10a has been reassigned as genotype 3, subtype k. Genotypes 7, 8, 9, and 11, belonging to clade 6, have been reassigned to genotype 6, subtypes c to l (25, 26, 27). These six HCV genotypes have different geographical distributions (21, 30, 32).

Treatment options for chronic HCV infections are poor. At the moment, the only accepted antiviral therapy with proven effectiveness is a combination therapy of (peg)interferon alpha and ribavirin. The overall success rate of this antiviral treatment ranges from 50% to 90% (11). According to a National Institutes of Health (NIH) 2002 panel, several factors are associated with successful treatment response, including lower baseline HCV RNA levels, lower fibrosis and inflammation scores upon liver biopsy, lower body weight, and lower body surface area, but the most important predicting factor is HCV genotype (24). Patients infected with HCV genotype 1 respond least to therapy, while patients infected with genotypes 2 and 3 show the best responses (14, 17, 22). For HCV genotypes 4, 5, and 6, treatment data are scarce, but it is recommended to treat these individuals using the same regimen as for patients infected with genotype 1 (7, 13, 18, 31). Nearly all patients experience side effects with the antiviral therapy. These side effects can be severe and contribute to discontinuation rates of 10 to 14% and dose reductions for 7 to 42% of patients, depending on the type and length of treatment (16). Therefore, it is important that clinicians have the appropriate information to make individual treatment choices in order to maximize the chance of successful treatment outcome for each individual patient, rendering HCV genotyping assays important and useful tools to optimize treatment type, duration, and dose.

In this paper, we evaluate Versant HCV genotype assay (LiPA) 2.0 (CE marked in Europe; for research use only; not for use in diagnostic procedures in the United States) (manufactured by Innogenetics, distributed by Siemens Healthcare Diagnostics), which uses sequence information from the core region in addition to sequence information from the 5' untranslated region (5'UTR), allowing an improved and more accurate distinction between HCV genotype 1 and subtypes c to l of genotype 6 and between subtypes a and b of genotype 1.

RESULTS

Clinical accuracy study. Table 1 summarizes the results for the 326 specimens that were used for the reference method comparison. Upon initial testing, 93.3% (304/326) of the specimens gave interpretable genotype results, 2.1% (7/326) failed to amplify, and 4.6% (15/326) amplified but gave uninterpretable results. Of the 304 specimens that yielded a genotype result, 99.3% (302/304) gave results that agreed with the reference method. After specimens that yielded no genotype result were retested, 96.0% (313/326) of the specimens gave interpretable genotype results, 3.5% (12/326) amplified but remained uninterpretable, and 0.3% (1/326) failed to amplify. Of the 313 specimens that yielded a genotype result after repeat testing, 99.4% (311/313) gave results that agreed with the reference method. Table 2 shows that the specimens that did not amplify or give interpretable results were distributed across ge notypes. The two specimens that initially gave results that disagreed with those obtained by the reference method were retested, and both gave retest results that agreed with those obtained by the reference method.

In order to determine the core amplification efficiency, 156 genotype 1 and genotype 6 (c to l) samples were analyzed. Two samples showed negative AMPL CTRL 1 lines and were excluded from further analysis. Of the remaining 154 genotype 1 and genotype 6 (c to l) samples, 1 sample had a negative AMPL CTRL 2, r esulting in the amplification of 99.4% (153/154) samples.

The clinical subtype efficiency for HCV genotypes 1a and 1b was determined using 129 samples that were genotype 1a or 1b based on reference sequencing and genotype 1, 1a, or 1b based on LiPA genotyping; this determination was based on initial testing only, excluding repeat testing of initial amplification failures and uninterpretable results. Three out of 129 samples were indeterminate at the subtype level, resulting in a clinical HCV genotype 1 subtype efficiency of 97.7% after initial testing. Upon repeat testing, all samples gave a correct consensus subtype result. All of the 126 samples that were genotype 1a or 1b by LiPA were concordant with sequencing.

In order to check whether Versant HCV genotype assay (LiPA) 2.0 was able to determine the correct genotype for samples with viral loads at the upper limit of detection, 22 samples with viral loads ranging from 4.0 x 106 IU/ml to 8.7 x 106 IU/ml were selected, and the genotype success rate and the percentage of agreement with the reference method for these high-concentration specimens were estimated. For all these samples, Versant HCV genotype assay (LiPA) 2.0 produced the same genotype results as the genotype result determined by NS5b sequencing and phylogenetic analysis, resulting in both a genotype success rate and an agreement with the reference method of 100%.

Reproducibility study. Table 3 summarizes the valid, indeterminate, correct, and incorrect genotype results for each reproducibility panel member. In total, 3.3% (16/486) of reactions gave indeterminate results (defined as specimens with either an amplification failure or an uninterpretable result) and 96.7% (LCL, 95.0%) yielded an interpretable genotype result. Of the 470 specimens with interpretable results, 100% (LCL, 99.4%) gave the correct genotype. The indeterminate results occurred at all sites, with all three reagent lots, and in multiple assay runs. There were no significant performance differences seen for the Versant HCV genotype assay (LiPA) 2.0 system across sites/operators (P = 0.43) or across reagent lots (P = 0.88). The genotype success rates at the individual sites were 98.1% for site 1, 97.6% for site 2, and 94.4% for site 3. The genotype success rates for the individual lots were 96.9% using lot 1, 97.5% using lot 2, and 95.7% using lot 3.

Comparison study. Table 4 gives an overview of the results of the comparison study after original testing and after repeat testing. Of the 100 specimens tested, 13 specimens initially produced uninterpretable results by either Versant HCV genotype assay (LiPA) 1.0 or Versant HCV genotype assay (LiPA) 2.0 or both assays. The HCV RNA concentrations of these 13 samples ranged from 14,615 to 2,500,000 IU/ml. These specimens were retested using both ass ays. After repeat testing, three specimens remained uninterpretable by both versions of the assay, five remained uninterpretable by Versant HCV genotype assay (LiPA) 1.0, and one remained uninterpretable by Versant HCV genotype assay (LiPA) 2.0. For all six specimens that gave a genotype result by only one version of the assay, the observed genotype result agreed with that obtained by sequencing the NS5b region of the HCV genome. After repeat testing, 83 specimens were concordant by both assays at the genotype level. Of these, 16 specimens had concordant genotypes by both assays, but one of the assays failed to give a subtype, resulting in a total of 67 concordant specimens when the subtype level is taken into account. Results from eight specimens were discordant between the two assays, and results from nine specimens were uninterpretable by at least one of the assays. The total number of interpretable specimens by both assays was 91, of which 83 had concordant results at the genotype level only (91.2%; LCL, 84.7%). Table 5 shows the number o f genotype and subtype results produced by both assays for the 100 specimens tested after repeat testing.

Phylogenetic analysis of a coding region, or even more, the complete genome, is considered the gold standard for identifying different HCV genotypes (6). However, since this method is expensive and time-consuming, it is impractical for large-scale genotyping projects (8). For this reason, commercial genotyping kits were developed for routine determination of HCV genotypes. Most commercially available HCV genotyping assays, including Versant HCV genotype assay (LiPA) 1.0, use the 5'UTR, since this region is highly conserved and therefore well suited for the development of detection methods. The reliability of genotyping methods highly depends on the amount of information (i.e., the number of informative sites) that is utilized for the discrimination of genetic variants. The 5'UTR is sufficiently variable for discrimination of HCV genotypes 1 to 5 and most subtypes of HCV genotype 6 (12, 28, 29, 32). However, it does not allow discrimination of HCV genotype 6 subtypes c to l from HCV genotype 1 and has only a limited subtyping accuracy (5, 29). To overcome the limitations of the 5'UTR, a new assay which uses additional sequence information from the core region of the HCV genome, Versant HCV genotype assay (LiPA) 2.0, has recently been developed (20). In this study, we evaluated the new assay and compared it with the previous version of the assay.

Our results indicate that Versant HCV genotype assay (LiPA) 2.0 yielded an interpretable genotype result for 96.0% of the samples and that 99.4% of the interpretable results agreed with the reference method, rendering it an accurate and reliable assay suitable for large-scale genotyping. This new assay outperforms the previous version of the line probe assay, since Versant HCV genotype assay (LiPA) 1.0 has an overall accuracy of 74%, taking subtype information into account (8, 23).

In the comparison study, eight specimens showed discordant results when tested with both assays. The NS5b sequencing results for these samples showed that Versant HCV genotype assay (LiPA) 2.0 gave the correct HCV genotype and subtype and thereby showed an improvement in identifying HCV-positive samples which are subtypes c to l of genotype 6 and in identifying the correct subtype of genotype 1. This improvement can be attributed to the additional information available from the core region of the HCV genome, which can better distinguish between genotype 1 and subtypes c to l of genotype 6 and between subtype a and b of genotype 1. This core information is not available in Versant HCV genotype assay (LiPA) 1.0, and this can lead to misinterpretation. For example, in a study by Chinchai et al., this assay could not discriminate HCV genotype 6a variants from HCV genotype 1b, and two samples found to be genotype 1 by the assay contained genotype 3 core sequences (5). Chen and Weck showed that Versant HCV genotype assay (LiPA) 1.0 cannot accurately distinguish HCV genotypes 1a and 1b, since in most cases, the 5'UTR is not heterogeneous enough for use in determining the HCV subtype (4). Several other studies report on moderate distinction at the subtype level (1, 2, 9, 12, 19). This is not surprising, since the 5'UTR is the most highly conserved region of the HCV genome, and only one or two nucleotide changes distinguish unique subtypes. Assigning correct genotypes and subtypes to HCV specimens is important for several research purposes, including epidemiological, phylogenetic, and natural history studies. Some studies even report that there is a slight difference in treatment outcomes between HCV genotype 1a- and HCV genotype 1b-infected patients, showing that correct subtype assignment is indispensable (3, 10, 30).

In conclusion, Versant HCV genotype assay (LiPA) 2.0 provides a rapid, sensitive, and accurate means of HCV genotyping and can be used as a routine tool to distinguish between the different HCV genotypes and subtypes. Considering the importance of genotype determination in understanding the epidemiology of the virus and in the management of hepatitis C treatment strategies, efficient genotyping tools are indispensable in clinical diagnostic settings.

Merck announced an expansion of the number of drugs it offers under its Merck Helps programs, including the Merck Patient Assistant program, the Merck Vaccine Patient Assistance program, the ACT program for cancer and hepatitis C drugs, and the SUPPORT program for HIV and AIDS drugs. The program provides medicines and vaccines free of charge to eligible patients, primarily the uninsured, who make up to 400% of the federal poverty level and can’t afford Merck drugs without assistance.

“Merck has historically recognized the critical need for people to have access to the prescription medicines and vaccines they require, even if they lose their insurance,” Merck EVP and chief medical officer Michael Rosenblatt said. “Our patient assistance programs now provide access to even more medicines for chronic conditions like asthma, diabetes and high blood pressure, allowing us to reach more people in need.”

Merck also said that its philanthropic arm, the Merck Company Foundation, made a grant to NeedyMeds, a nonprofit group that helps people who can’t afford medicines or healthcare costs by making information about assistance programs available. NeedyMeds plans to use the grant to translate its website into Spanish.

HIV-positive women were no more likely to have a bone fracture than HIV-negative women, according to a study published online September 20 in AIDS. The new data run counter to a growing concern that HIV might be causing age-related problems, including bone problems, to occur at a younger age in HIV-positive people.

As the population of people with HIV grows older, the research community is increasingly turning its focus toward diseases and conditions that typically strike the elderly, including cardiovascular disease, certain cancers and bone problems.

Numerous studies have found that people with HIV, men and women alike, often have poorer bone health than their HIV-negative counterparts. What isn’t certain, however, is whether the reduced levels of bone density found in HIV-positive people are resulting in an increase in bone fractures. While one large study did find an increase in fractures of the hip, spine and wrist, it could not account for many bone fracture risk factors, such as body mass, smoking status and hepatitis C virus (HCV) infection.

To further define the actual risk for fractures in HIV-positive women, Michael Yin, MD, from Columbia University Medical Center in New York City, and his colleagues analyzed data from the Women’s Interagency HIV Study (WIHS)—a large cohort study that has been following more than 3,000 HIV-positive and HIV-negative women since at least 2001. This analysis included 1,728 HIV-positive women and 663 HIV-negative women.

Most of the women in both groups were black or Latina and tended to be a bit overweight. About half were smokers, and roughly 20 percent in both groups were diabetic. The groups differed on several counts, however. HIV-positive women tended to be older, to have begun menopause and to also be infected with HCV.

The rate of new fractures was similar between the groups, with 148 HIV-positive women (8.6 percent) and 47 HIV-negative women (7.1 percent) experiencing a new fracture during an average follow-up of 5.4 years.

Yin’s team found that when they accounted for known risk factors—such as smoking, HCV status and the women’s ratio of height to body weight (body mass index or BMI)—HIV-positive women were no more likely to experience a fracture than HIV-negative women. In fact, the only factors associated with bone fracture risk overall were: older age, white race, HCV status and high kidney protein (serum creatinine) levels.

When Yin and his colleagues restricted their analysis only to HIV-positive women, they found that smoking history, opiate-use history and onset of menopause were all associated with a higher fracture risk. Of interest, low CD4 counts were not associated with an increased fracture risk among HIV-positive women, nor was any class of antiretrovirals (ARVs) including tenofovir (found in Viread, Truvada and Atripla), a commonly used HIV medication that has been tied to decreased bone mineral density.

The authors caution that their study results can’t be applied to older and post-menopausal women, but they concluded, “Our data provide some reassurance that fracture risk is modest in predominantly premenopausal HIV-infected women.”

“However, further research is necessary,” they continued, “to assess fracture risk as these women transition through menopause and to clarify whether fracture risk differs among antiretroviral regimens.”

WASHINGTON—Elderly chimpanzees living at a nonresearch facility in Alamogordo, N.M., should not be shipped to Texas for use in invasive experiments, says a federal complaint to be filed Sept. 23 with Kathleen Sebelius, secretary of Health and Human Services.

Doctors and scientists with the Physicians Committee for Responsible Medicine (PCRM) seek to halt the National Institutes of Health’s planned transfer of nearly 200 federally owned chimpanzees to a laboratory in San Antonio, Texas. New Mexico Gov. Bill Richardson and primatologist Jane Goodall have also spoken out against the proposed transfer.

The doctors’ legal petition invokes the Chimpanzee Health Improvement Maintenance and Protection (CHIMP) Act, enacted to ensure that chimpanzees used in experiments for many years are retired to sanctuaries. Many Alamogordo chimpanzees are elderly, have been used repeatedly for invasive procedures, and deserve a peaceful retirement, PCRM’s complaint says. For example, Flo, Guy, and James were born in 1957, 1959, and 1960, respectively. Many suffer from heart disease, making them especially unsuitable for medical experiments.

“There can be no scientific, legal, or ethical justification for returning a 50-year-old chimpanzee to laboratory experiments,” says John J. Pippin, M.D., F.A.C.C., senior medical and research adviser for PCRM. “More than five decades of experiments have shown us that chimpanzees are poor models for researching human diseases. Are we such slow learners that we now return to these outdated methods?” In submitting the petition to Secretary Sebelius, Dr. Pippin is joined by 11 other authorities, including Harvard professor Richard Wrangham, Ph.D., and University of New Mexico professor John Gluck, Ph.D.

The doctors and scientists argue in their complaint, “Chimpanzees have repeatedly proved to be poor models for human disease research, including for HIV—a disease for which repeated failures have led most researchers to stop using chimpanzees—as well as for hepatitis, malaria, and cancer. Therefore, these animals are not necessary for this type of research. Superior alternatives are available and researchers continue to develop new, cutting-edge models for human disease research.” The hepatitis C virus behaves very differently in humans and chimpanzees, and decades of experiments have failed to produce a human vaccine. Leading hepatitis C researchers are using human-cell-based research methods.

The mothers of some Alamogordo chimpanzees now live in a sanctuary in Washington State—Chimpanzee Sanctuary Northwest—and have come to the attention of Sen. Maria Cantwell. Sen. Cantwell recently introduced the Great Ape Protection Act (GAPA), S. 3694, which would advance medical research by phasing out wasteful and misleading chimpanzee experiments and releasing federally owned chimpanzees to sanctuaries. A parallel House bill, H.R. 1326, has gained significant momentum and now has 149 co-sponsors. The United States is the last country in the world that permits and funds large-scale chimpanzee research and testing. Earlier this month, the European Union banned great ape experiments.

Founded in 1985, the Physicians Committee for Responsible Medicine is a nonprofit health organization that promotes preventive medicine, conducts clinical research, and encourages higher standards for ethics and effectiveness in research.

ScienceDaily (Sep. 22, 2010) — Researchers at Cedars-Sinai Medical Center and 50 other centers found that weight-based dosing of taribavirin reduces rates of anemia while increasing sustained virologic response (SVR) in patients with chronic hepatitis C (HCV). Full details of this study are available in the October issue of Hepatology, a journal published by Wiley-Blackwell on behalf of the American Association for the Study of Liver Diseases (AASLD).

Chronic HCV is typically treated with ribavirin (RBV). When used in combination with peginterferon alfa (peg-IFN), RBV significantly enhances on-treatment virologic response and reduces relapse. However, RBV, particularly the combination of interferon and RBV, is associated with hemolytic anemia, a significant toxicity resulting from the accumulation of RBV in red blood cells. Taribavirin (TBV), formerly known as viramidine, is a nucleoside analog and oral pro-drug of RBV that is less able to enter red blood cells, and should therefore be associated with significantly less anemia.

This theory was demonstrated in two previous phase 3 trials. While statistically less anemia was observed in patients treated with TBV compared to RBV, the primary efficacy endpoint of these studies, a non-inferior SVR between the TBV and RBV, was not achieved. Detailed subgroup analyses of the data suggest fixed dosing as opposed to weight-based dosing, and the selection of an inadequate dose, are to blame. The present multi-center study explored several higher weight-based doses of TBV to determine a dosage regimen that was able to deliver comparable responses to RBV with fewer incidences of anemia.

A phase 2b randomized, open-label, active-controlled, parallel-group study was conducted in 278 treatment-naïve, genotype 1 patients stratified by body weight and baseline viral load at 51 centers in the United States between March 2007 and October 2008. Patients were randomized 1:1:1:1 to receive TBV (20, 25, or 30 mg/kg/day) or RBV (800 -1400 mg/day) with pegylated interferon alfa-2b for 48 weeks.

The primary efficacy endpoint was early virologic response (EVR) defined as the proportion of patients with at least a 2-log decrease from baseline in serum HCV RNA levels at treatment week 12. Additional efficacy endpoints included SVR, undetectable HCV RNA at treatment weeks 4, 24 and 48, and viral relapse for those who were responders at the end of treatment. A total of 86 (41%) of TBV patients and 25 (36%) of the RBV group completed treatment and follow up. The most commonly cited reasons for premature withdrawal were lack of response (29%) and adverse events (20%).

The present study demonstrated that weight-based dosing of TBV achieved comparable efficacy to RBV as demonstrated by SVR. This was observed in all three TBV weight-based dose treatment groups, which met the study's primary end-point. Patients treated with TBV had less than half the anemia compared to RBV treated patients. These results suggest weight-based dosing of TBV can significantly improve the tolerability of HCV treatment while maintaining efficacy. Specifically, the 25 mg/kg dose offered the optimal balance of efficacy and safety in this patient population.

Notably, fewer patients treated with TBV required dose reductions (13-28%) compared to 32% of patients treated with RBV. Less frequent dose modification in patients treated with TBV may alleviate the need to utilize erythropoiesis-stimulating agents (ESAs). Several studies have demonstrated the use of ESAs can significantly decrease the need to dose reduce RBV and leads to an improvement in the quality of life during HCV treatment, but fails to improve the SVR. The use of ESAs also adds significant cost to HCV treatment and is associated with serious adverse events including thrombosis and red cell aplasia.

Lead investigator Dr. Fred Poordad concludes, "These data suggest TBV may be an effective agent to substitute for RBV in the future and could be incorporated in upcoming trials utilizing emerging small molecules for HCV treatment."

Editorial author Dr. Paul Kwo comments, "If TBV can be shown to preserve or improve efficacy rates in combination with direct-acting antiviral agents (DAAs) and Peg IFN, with lower rates of anemia, the use of TBV in these clinical settings would be a welcome addition to the HCV armamentarium as we begin to expand the HCV populations that we treat. TBV may have a role in populations particularly sensitive to ribavirin-related anemia. However, with the commencement of several trials comprising of multiple combinations of DAAs with and without pegIFN/RBV, and the development of newer protease inhibitors with potentially lower rates of anemia, the role of TBV remains less precisely defined and could potentially have a finite life cycle."

22nd September 2010 - The NHS regulator, NICE, has updated guidance on the treatment of hepatitis C. This covers the use of peginterferon alfa (2a or 2b) and ribavirin for the treatment of chronic hepatitis C.

This new guidance reflects changes in licensing and recommends their wider use and, where appropriate, shorter treatments for adults with the disease.

Hepatitis C

Estimates from the Health Protection Agency suggest that approximately 142,000 people between the ages of 15-59 years had chronic hepatitis C (HCV) in England and Wales in 2003. More than 90% of all newly diagnosed infections in the UK occur in injecting drug users.

People infected with HCV often don’t have any symptoms, but about 20% will develop acute hepatitis and will experience symptoms such as malaise, weakness and anorexia. About 80% of those infected with the virus go on to develop chronic hepatitis.

The rate of progression from mild to severe disease is slow, taking about 20-50 years from the time of infection. About 30% of infected people develop cirrhosis within 20-30 years, and some of these people are at a high risk of developing hepatocellular carcinoma. Some people with end-stage liver disease or hepatocellular carcinoma may require liver transplants.

Revised guidance

The National Institute for Health and Clinical Excellence (NICE) guidance advises that:

Combination therapy with peginterferon alfa (2a or 2b) and ribavirin is recommended as a treatment option for adults with chronic hepatitis C who have been treated previously with peginterferon alfa (2a or 2b) and ribavirin in combination; or with peginterferon alfa monotherapy, and whose condition either did not respond to treatment, or responded initially to treatment but then relapsed; or for adults who are also infected with HIV

Shortened courses of combination therapy with peginterferon alfa (2a or 2b) and ribavirin are recommended for the treatment of adults with chronic hepatitis C who have a rapid virological response to treatment at week 4 that is identified by a highly sensitive test, and who are considered suitable for a shortened course of treatment.

Many people ‘have no idea they have it’

A recent All Party Parliamentary Hepatology Group report on hepatitis C identified the disease as being one of the main reasons for the large rise in the number of people dying from liver disease. The report estimates that there could be as many as 466,000 people living with hepatitis C in the UK.

Sir Andrew Dillon, NICE Chief Executive, says in a statement: “Many people exposed to the disease have no idea they have it since it can remain symptomless for many years. However, hepatitis C is a potentially debilitating condition, and about 80% of those with the virus go on to develop chronic hepatitis, sometimes as long as 50 years after they were first infected. By widening access to these drugs, this guidance will give clinicians and people living with hepatitis C more treatment options.”

HOERSHOLM, Denmark and SAN DIEGO, September 22, 2010 /PRNewswire/ -- Santaris Pharma A/S, a clinical-stage biopharmaceutical company focused on the discovery and development of RNA-targeted therapies, today announced that it has advanced miravirsen (SPC3649), the first microRNA-targeted drug to enter clinical trials, into Phase 2 studies to assess the safety and tolerability of the drug in treatment-naive patients infected with the Hepatitis C virus (HCV).

Paving the way to conduct the first clinical trials of a microRNA-targeted drug in the United States, Santaris Pharma A/S also received acceptance of its Investigational New Drug (IND) application from the U.S. Food and Drug Administration (FDA). In addition to the United States, the Phase 2a clinical trials will be conducted in the Netherlands, Germany, Poland, Romania, and Slovakia.

The World Health Organization estimates about 3% of the world's population has been infected with HCV and that some 170 million are chronic carriers at risk of developing liver cirrhosis and/or liver cancer(2). Approximately 3-4 million Americans are chronically infected with an estimated 40,000 new infections per year(1). In Europe, there are about 4 million carriers(2). The current standard of care, pegylated interferon in combination with ribavirin, is effective in only about 50% of those treated(1).

Developed using Santaris Pharma A/S proprietary Locked Nucleic Acid (LNA) Drug Platform, miravirsen is a specific inhibitor of miR-122, a liver specific microRNA that the Hepatitis C virus requires for replication. Miravirsen is designed to recognize and sequester miR-122, making it unavailable to the Hepatitis C virus. As a result, the replication of the virus is effectively inhibited and the level of Hepatitis C virus is reduced.

"Advancing miravirsen, the first microRNA-targeted drug to enter clinical trials, into Phase 2 studies in patients with Hepatitis C demonstrates Santaris Pharma A/S leadership in developing RNA-targeted medicines," said Arthur A. Levin, Ph.D., Vice President, Chief Development Officer and President, US Operations. "Receiving IND acceptance from the FDA to conduct the first clinical trials with a microRNA-targeted drug in the United States brings Santaris Pharma A/S one step closer to potentially providing a growing number of patients chronically infected with HCV with a more effective and better tolerated treatment option."

The LNA Drug Platform is the only technology with both mRNA and microRNA targeted drugs in clinical trials, reinforcing the broad utility of the platform. The unique combination of small size and very high affinity, which is only achievable with LNA-based drugs, allows this new class of drugs to potently and specifically inhibit RNA targets in many different tissues without the need for complex delivery vehicles. LNA-based drugs are a promising new type of therapy that enables scientists to develop drugs to attack previously inaccessible pathways.

"Using our LNA Drug Platform to advance the first microRNA-targeted therapy into human clinical trials was certainly a scientific breakthrough," said Henrik Oerum, Ph.D., Vice President and Chief Scientific Officer of Santaris Pharma A/S. "We are extremely pleased with the results of the Phase I trials and excited to progress miravirsen into Phase 2 clinical trials. Because of its unique mechanism of action and tolerability profile, miravirsen has the potential to be an effective treatment option for patients with HCV."

The randomized, double-blind, placebo-controlled, ascending multiple-dose Phase 2a study will assess the safety and tolerability of miravirsen and is designed to enroll up to 55 treatment-naïve patients with chronic Hepatitis C virus genotype 1 infection. Secondary endpoints include pharmacokinetics of miravirsen and its effect on viral load. Miravirsen will be given as subcutaneous injections weekly or every other week for four weeks.

Data from Phase 1 clinical studies with miravirsen in healthy volunteers show that the drug is well tolerated. A recent study published in Science demonstrated that miravirsen successfully inhibited miR-122 and dramatically reduced Hepatitis C virus in the liver and in the bloodstream in chimpanzees chronically infected with the Hepatitis C virus(3). Miravirsen provided continued efficacy in the animals up to several months after the treatment period with no adverse events and no evidence of viral rebound or resistance.

MicroRNAs have emerged as an important class of small RNAs encoded in the genome. They act to control the expression of sets of genes and entire pathways and are thus thought of as master regulators of gene expression. Recent studies have demonstrated that microRNAs are associated with many disease processes. Because they are single molecular entities that dictate the expression of fundamental regulatory pathways, microRNAs represent potential drug targets for controlling many biologic and disease processes.

About Locked Nucleic Acid (LNA) Drug Platform

The LNA Drug Platform and Drug Discovery Engine developed by Santaris Pharma A/S combines the Company's proprietary LNA chemistry with its highly specialized and targeted drug development capabilities to rapidly deliver potent single-stranded LNA-based drug candidates against RNA targets, both mRNA and microRNA, for a range of diseases including metabolic disorders, infectious and inflammatory diseases, cancer and rare genetic disorders. The LNA Drug Platform overcomes the limitations of earlier antisense and siRNA technologies to deliver potent single-stranded LNA-based drug candidates across a multitude of disease states. The unique combination of small size and very high affinity, which is only achievable with LNA-based drugs, allows this new class of drugs to potently and specifically inhibit RNA targets in many different tissues without the need for complex delivery vehicles. LNA-based drugs are a promising new type of therapy that enables scientists to develop drugs to attack previously inaccessible clinical pathways. The most important features of LNA-based drugs include excellent specificity, providing optimal targeting; increased affinity to targets providing improved potency; and strong pharmacology upon systemic delivery without complicated delivery vehicles.

About Santaris Pharma A/S

Santaris Pharma A/S is a privately held clinical-stage biopharmaceutical company focused on the discovery and development of RNA-targeted therapies. The Locked Nucleic Acid (LNA) Drug Platform and Drug Discovery Engine developed by Santaris Pharma A/S combine the Company's proprietary LNA chemistry with its highly specialized and targeted drug development capabilities to rapidly deliver potent single-stranded LNA-based drug candidates across a multitude of disease states. The Company's research and development activities focus on infectious diseases and metabolic disorders, while partnerships with major pharmaceutical companies include a range of therapeutic areas including cancer, cardiovascular disease, infectious and inflammatory diseases, and rare genetic disorders. The Company has strategic partnerships with miRagen Therapeutics, Shire plc, Pfizer, GlaxoSmithKline, and Enzon Pharmaceuticals. As part of its broad patent estate, the Company holds exclusive worldwide rights to all therapeutic uses of LNA. Santaris Pharma A/S, founded in 2003, is headquartered in Denmark with operations in the United States. Please visit http://www.santaris.com/ for more information.

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